Volume 9, Issue 2, February 2024
ISSN No:-2456-2165
Impact of Quercetin and Omega-3 Fatty-Acid on
Lead-Induced Alterations in Reproductive
Parameters in Male Wistar Rats
Nwaokocha SC1; Gekpe CG2; Ezeani Chidiebere3; Ofem OE.4* (Professor)
Department of Physiology, Faculty of Basic Medical Sciences, University of Calabar, Calabar, Nigeria
Correspondence Author:- Ofem OE.4*
Abstract:- Exposure to environmental toxins like Lead
has been associated with male infertility. Whether or not
potent antioxidants like omega 3 Fatty acids and/or
quercetin could reduce the effect of lead on male sexual
functions deserve scientific investigation. Thirty-five (35)
male albino Wistar rats were assigned into 7 groups:
Group 1 (normal control), Group 2 (sham-control1),
Group 3 (sham-control 2), Group 4 (Lead group), Group
5 (Lead + Omega-3), Group 6 (Lead + Quercetin) and
Group 7 (Lead + Omega-3 + Quercetin). Lead was given
orally at 20mg/kg bwt, quercetin 20mg/kg bwt s.c,
Omega-3 14.29mg/kg bwt orally. The animals all had
free access to rat food and water for 56 days. After which
they were sacrificed, and semen and blood samples were
collected for assay. The results showed no significant
difference between normal control group and sham
controls. Sperm function parameters (sperm count,
motile, viable and normal sperms) in the lead treated
group was significantly reduced compared to the control.
Omega 3 and/or quercetin administration reversed the
reductions in sperm function parameters to near control
levels. Hormone levels (LH and Testosterone) were
significantly reduced in lead group compared to the
normal control but were reversed after quercetin and
omega 3 treatment. However, there was no significant
difference in the FSH level among experimental groups.
Conclusively, omega 3 and/or quercetin ameliorates the
harmful effects of lead on reproductive parameters by
improving sperm functions (total sperm count, viability,
motility, and morphology). A combination of both
quercetin and omega 3 provided better ameliorative
effect than either omega 3 or quercetin.
Keywords:- Infertility, Quercetin, Omega 3, Lead, Sex
Hormones, Rats.
I. INTRODUCTION
The International Committee Monitoring Assisted
Reproductive Technologies (ICMART) and World Health
Organisation define clinical infertility as the inability of
pregnancy to occur after 12 months of regular unprotected
sexual intercourse, where either one or both parties
contribute to the challenge. When a sexually mature male is
unable to impregnate a sexually mature female, it is
described as male infertility. This accounts for This accounts
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for 1/3rd (20-30%) of infertility cases [1]. Male infertility is
mostly associated with difficulty in ejaculation, small
volumes of semen ejaculated, absence or low sperm levels,
abnormally shaped (morphology) sperm and abnormal
movements (motility), erectile dysfunction (impotency) [2].
Also, exposure to environmental elements such as heat,
toxins, and industrial chemicals, heavy metals like lead,
radiation and unhealthy lifestyles affect sperm production
and function. Male infertility can be diagnosed via
laboratory semen analysis, thereby testing for, sperm
production levels sperm count, sperm functionality
(morphology and motility) and sperm concentration, and
assay of male sex hormones. Lead is a neurotoxin that easily
accumulates in soft tissues and bones and can damage a lot
of body systems. Lead toxicity affects almost every body
function. [3]. Lead toxicity has a devastating effect on body
system and is fast become a serious environmental disease.
Lead is highly persistent in environment especially in
developing countries where its use is still high [4].
Plant pigments (flavonoids) such as quercetin can be
found in a lot of plants and food. Quercetin has antioxidant
and anti-inflammatory effects that reduce swellings, kill
cancer and control body parameters, making it fit as a potent
disease resistant and improves overall health [5]. Quercetin is
ubiquitous in nature, available in many fruits such as apple,
vegetables, berries and tomatoes.
Aside other health benefits, studies have shown that
quercetin improves male fertility (sperm motility) from
leukocytospermic patients and protect oxidative damage to
sperm [6].
Omega 3 fatty acids are a subclass of polyunsaturated
fatty acids (PUFAs). They have a double bond and three
atoms from their terminal methyl group. They are ubiquitous
in nature and is an important constituents of animal lipid
metabolism [7]. Omega 3 oils play very important roles in
human physiology and diet. Although they are not
synthesized in the body, they are obtained from diet.
Docosahexaenoic acid (DHA) is a major constituent of
omega-3 that is found in male reproductive organs and the
brain. Animal studies has shown that in early reproductive
events, omega 3 acids play important roles, can restore
fertility and improve spermatogenesis in male rodents [8, 9].
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ISSN No:-2456-2165
Some other studies showed misshaped sperm due to DHA
absence resulting in infertility in Male Mice [10].
Lead exposure in both men and women is popular for
many adverse reproductive effects [11, 12]. Some of these
harmful effects for males are: infertility, changes in serum
testosterone, reduction in libido, abnormal prostatic
functions, and alteration in spermatogenesis [13, 14].
This research was therefore aimed at investigating the
effect of a combination of quercetin and omega 3 to curb the
effect of lead on reproductive parameters in male rats.
II. MATERIALS AND METHODS
 Laboratory Animals
Thirty-five (35) male albino Wister rats weighting
between 180-220g were obtained from the animal house of
the Department of Physiology, University of Calabar,
Calabar, Nigeria for the study. All rats had access to free
water and chow. The animals were acclimatized for one
week, Ethical approval was obtained from the Animal
Research Ethics Committee of Faculty of Basic Medical
Sciences, University of Calabar, (approval number:
231PHY2523).
 Experimental Design and Drug Administration
The animals were weighed and distributed into seven
groups of five animals. Lead was administered at a dose of
20mg/kg body weight.
Omega-3 was administered at a dose of 14.29 mg/kg
(extrapolated from human dose of 1000 mg / 70 kg) was
administered once daily by dissolving 1 capsule in 5mL of
Olive oil and 0.01mL was given to 100g rat15.
A dose of 20mg/kg body weight of quercetin was
administered to the rats subcutaneously once daily. 0.1g of
quercetin was dissolve in 5ml of 2% dimethly sulfoxide
(DMSO) solvent, then given at 0.1ml/100g body weight,
subcutaneously and once daily.
 Group 1 (control): Received normal rat chow + drinking
water
 Group 2 (Sham control-1): DMSO (2%) 1mL/kg orally
and once daily.
 Group 3 (Sham control-2): Olive oil (0.1 mL/kg orally
and once daily)
 Group 4: (Lead group): Lead (20 mg/kg, o.p)
 Group 5: (Lead + Omega-3): Lead (20 mg/kg, o.p) +
Omega-3 (14.29 g/kg o.p)
 Group 6: (Lead + Quercetin): Took Lead (20 mg/kg, o.p)
+ Quercetin (20 mg/kg s.c)
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 Group 7: (Lead + Quercetin + Omega-3): Lead (20
mg/kg, o.p) + Omega-3 (14.29mg/kg o.p) + Quercetin
(20mg/kg, subcutaneously)
All the animals had free access to normal rat feed and
drinking water. The feeding regimens lasted for 56 days.
The animals were euthanized. The feeding regimens lasted
56 days (8 weeks). The animals were then fasted overnight,
weighed and anaesthetized with 5% chloroform. Blood
samples were collected via cardiac puncture16 into plain
caped sample bottles, then left to stand for 2 hours to clot,
the blood was then centrifuged and serum extracted from the
supernatant. Semen samples were also collected from the
epididymis into 9% NaCl solution in sample bottles for
semen analysis.
 Semen analysis
 Determination of Sperm Count
The epididymal content was obtained with forcept,
weighed and placed on a petri-dish containing physiological
saline. The suspension was separated into fragments by
fattening through 80 micronmeter stainless mesh. A tissue –
free aliquot obtained was loaded into the Neubauer
haemocytometer (deep 1/10 Labart. Germany). Different
sperm counts were done using microscope17.
 Mean Count for each Rat was Calculated using the
Formula: Sperm Count = (Total No. of Sperm cells in
the Cytometer) / (Mean Value)
 Determination of Sperm Viability
The improved one step eosin-nigrosin staining
technique was utilized18. A fraction of each suspension of
sperm sample was mixed with equal volume of eosin-
nigrosin stain. Smears were made on slides and air-dried.
The air dried smears were then prepared on a slide for each
sample. The slides were coated randomly and examine
under the microscope for viability. The percentage viability
was calculated based on the number of viable (live) sperm
cells divided by the number of sperm cells within 30
minutes multiply by 100.
 Determination of Sperm Motility and pH
Semen samples from different treated groups were
collected and dropped on a glass slide and viewed under the
(Olympus, Japan) microscope at x400 magnification. The
percentage of sperm was analyzed for progressive motile
sperm (PMS), non-progressive – motile sperm (NPMS), and
distinguished – by the movement of the sperm.
The pH meter was used to determine the pH of the
semen samples.
 Determination of Sperm Morphology
One drop of suspension of each semen sample was
placed on a glass slide. The slide was air dried and stained
with 1% eosin. The morphological abnormalities of the
sperm were evaluated from a total of two hundred sperm per
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ISSN No:-2456-2165
animal19. The results were recorded as percentage of the
abnormal sperms.
 Hormonal Assay
 Determination of Testosterone
ELISA kit was employed for the determination of
testosterone levels. It was carried out in Zone-3 diagnostic
laboratory, Akpandem Street, Calabar, Nigeria. A working
solution of the testosterone – HRP conjugate and wash
buffer were prepared. The required number of microwell
strips were displaced.100µl of the conjugate working
solution was placed in each well. This was followed by
addition of 50µl of each calibrator, control and specimen
sample into corresponding labelled wells. Duplicates of the
wells were made20.
All wells were incubated on a plate shaker
approximately 200rpm for 1hr at room temperature. Each
well was washed 3 times with 300µl wash buffer per well
and the plate was tap firmly against absorbent paper to
ensure that it was dry. Thereafter, 150µl of 5,5-
tetrameltylbenzidine (TMB) substrate was pipette into each
well at time intervals and re-incubated on a plate shaker 10 –
15 minutes at room temperature (or until calibrator attain a
dark) blue color for desired OD. At the expiration of the
incubation, 5µl of stop solution was added to each well at
the same time intervals. Finally, reading of the plates was on
a microwell plate reader at 450nm within 20 minutes.
 Determination of Luteinizing Hormone (LH) and Follicle
Stimulating Hormone (FSH)
ELISA kit was adopted for the determination of
luteinizing hormone (LH) and Follicle stimulating hormone.
LH- conjugate and wash buffer solutions were prepared.
25µl of each calibrator, control and specimen samples was
pipetted into correspondingly labelled wells in duplicate.
The plate shaker was incubated for 200rpm for 30 minutes at
room temperature. The wells were then incubated on the
plate shaker for 15 – 20 minutes at room temperature (or
until calibrator attain dark blue color for desired OD).
Thereafter, 50µl of stop solution was transferred into each
well at the same time interval. Reading of the plate on a
micro well plate reader was at 450nm within 20 minutes
after dilution of the stop solution 20.
Similar procedures were followed for the
determination of follicle stimulating hormone. However,
FSH binds to anti β-FSH receptors and required a volume of
300ml of Mela and 2 diluent buffers in the buffer bottle to
properly process the assay run20.
 Statistical Analysis
The data obtained were presented as mean ±SEM, data
were analyzed using one-way analysis of variance followed
with Tukey post hoc test. This was done with the aid of a
statistical package, IBM SPSS Version 25.0 for windows.
P<0.05 was considered significant.
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III. RESULTS
 Sperm Count, Motile, Viable Sperms and Sperm
Morphology in the Different Experimental Groups
The total sperm count (x106/L) for the control, sham-1
and sham-2 were 192.20 ±21.00, 211.80 ±23.11, and 196.20
±10.55 respectively. It was significantly (p<0.05) reduced in
the lead group 91.20 ±8.25 compared with other groups
(Lead + Omega 3, 170.60 ±16.68, Lead + quercetin, 150.60
±18.56 and Lead +quercetin + Omega 3, 185.40 ±17.22),
Table 1.
Percentage of motile sperm cells for the control was
88.00 ±2.55%, for the sham-1 it was 84.00 ±1.87%, for the
sham-2, 87.00 ±2.55%, lead group, 71.00 ±1.00%, Lead +
Omega 3, 78.00 ±1.22%, Lead + quercetin, 79.00 ±1.87%
and Lead +quercetin + Omega 3 it was, 80.00 ±2.74%. No
significant difference existed among the control groups. But
it decreased significantly (p<0.05) in the lead group
compared to the control and the treatment groups, Table 1.
The percentage of viable sperm cells was not
significantly different among the different experimental
groups. The control had a count of 90.60 ±, sham-1 (86.00
±) and sham-2 (87.40 ±), the lead, Lead + Omega 3, Lead +
quercetin and Lead +quercetin + Omega 3 had counts of
76.00 ±, 83.00 ±, 85.20 ± and 87.00± respectively, Table 1.
The percentage sperm cells with normal morphology
for the control different control groups were not
significantly different from each other, (control, 90.40
±0.40%, sham-1, 90.80 ±0.37% and sham-2, 88.00 ±1.22%).
It was significantly (p<0.05) lower in lead group (68.00
±3.74%) compared with the control groups and the other
treatment groups (Lead + Omega 3, 84.00 ±3.67%, Lead +
quercetin, 88.00 ±3.39% and Lead +quercetin + Omega-3,
89.00 ±2.92%), Table 1.
 Sex Hormones Concentration in the Different
Experimental Groups
There were no significant differences in concentrations
of follicle stimulating hormone (FSH) among the different
experimental groups. For the control FSH concentration was
8.26 ± 0.47, sham-1, 7.08 ± 0.27; sham-2, 7.50 ± 0.51; lead
group 7.12 ± 0.46; Lead + Omega 3, 6.28 ± 0.46; Lead +
quercetin it was 6.24 ± 0.14 and in the Lead +quercetin +
Omega 3 it was 6.98 ± 0.55, Fig. 1.
The luteinizing hormone (LH) levels (mIU/mL) for the
control groups were not significantly different from each
other. Control had LH level of 53.00 ± 0.89, sham-1, 48.80
± 2.01, and sham-2 49.20 ± 2.29. The lead group (16.00 ±
1.10) had significantly (p<0.05) lower LH concentration
compared with the control groups. The increase in LH
observed in Lead + Omega 3 (23.20 ± 0.49) and Lead +
quercetin (24.60 ± 0.68) were not significantly different
from the lead group, but Lead +quercetin + Omega 3 (33.00
± 3.10) had significantly higher LH levels compared with
lead group, Fig. 2.
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ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/IJISRT24FEB084

The Testosterone levels (ng/mL) for the control, sham-
1 and sham-2 groups were 36.00 ± 0.55, 33.20 ± 1.39 and
36.40 ± 3.59 respectively, showing no significant
differences among groups. It was significantly (p<0.05)
lower in the lead group (11.20 ± 0.49) compared with the
controls and other treatment groups. Values of testosterone
obtained in Lead + Omega 3 was 28.20 ± 0.73, the Lead +
quercetin had values of 26.80 ± 11.77 and in the Lead +
quercetin + Omega 3 it was 30.80 ± 1.02, Fig. 3.

Table 1 Comparison of Sperm Count, Motile, Viable Sperms and Sperm Morphology
Group Sperm count (x106/L) Motile sperm (%) Viable sperms (%) Normal morphology (%)
Control 192.20 88.00 90.60 90.40
±21.00 ±2.55 ±0.40 ±0.40

Sham fed 1 211.80 84.00 86.00 90.80

±23.11 ±1.87 ±4.58 ±0.37

Sham fed 2 196.20 87.00 87.40 88.00

±10.55 ±2.55 ±2.18 ±1.22

Lead 91.20 71.00 76.00 68.00

±8.25*ab ±1.00*ab ±2.45 ±3.74*ab

Lead + Omega 3 170.60 78.00 83.00 84.00

±16.68 ±1.22* ±3.39 ±3.67c

Lead + Quercetin 150.60 79.00 85.20 88.00

±18.56 ±1.87 ±4.31 ±3.39c

Lead + Omega-3 185.40 80.00 87.00 89.00

+ Quercetin ±17.22c ±2.74 ±3.39 ±2.92c
Values are Expressed as mean ±SEM, n = 5.
* = p<0.05 vs control
a = p<0.05 vs sham fed-1
b = p<0.05 vs sham fed-2
c = p<0.05 vs lead

Fig 2 Luteinizing Hormone Concentration in the Different

Experimental Groups
Values are Expressd as mean +SEM, n = 5.
Fig 1 Follicle Stimulating Hormone Concentration in the *= p<0.05 vs control
Different Experimental Groups a = p<0.05 vs sham fed-1
Values are Expressd as mean +SEM, n = 5. b = p<0.05 vs sham fed-2
No Significant Differences among Groups z = p<0.05 vs all groups

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Volume 9, Issue 2, February 2024
ISSN No:-2456-2165
Fig 3 Testosterone Concentration in the Different
Experimental Groups
Values are expressd as mean +SEM, n = 5
* = p<0.05 vs control
a = p<0.05 vs sham fed-1
b = p<0.05 vs sham fed-2
IV. DISCUSSION
When a sexually mature male is unable to impregnate a
sexually mature fertile female, it is described as male
infertility. This accounts 20-30% of infertility cases.1 Male
infertility is mostly associated with difficulty in ejaculation,
small volumes of semen ejaculated, absence or low sperm
levels, abnormally shaped (morphology) sperm and
abnormal movements (motility), erectile dysfunction
(impotency) [2]. Also, exposure to environmental elements
such as heat, toxins, and industrial chemicals, heavy metals
like lead, radiation and unhealthy lifestyles affect sperm
production and function. Lead exposure is known to cause
adverse health outcome in men and women [14]. Several
adverse reproductive outcomes have been reported to occur
in men exposed to lead. Some of which include; reduced
libido, effects on spermatogenesis, chromosomal damage,
infertility, abnormal prostatic function and changes in serum
testosterone [14]
This study was aimed at investigating the effect of a
combination of quercetin and omega 3 to curb the effect of
lead on reproductive parameters in male rats.
Results obtained indicated that in the lead group, there
was a significant decrease in sperm motility compared to the
control groups. This could be as a result of the direct effect
of lead on reproductive organs. There was an increase in the
motility of sperm in the Quercetin and Omega 3 treated
groups, suggesting that quercetin and omega 3 treatments
can improve sperm motility.
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Earlier reports showed that a supplementary treatment
with omega 3 fatty acids on infertile men caused a
significant improvement in sperm motility and plasma
concentration of DHA [21].
Also studies indicate that membrane integrity of sperm
was preserved to near normal following quercetin treatment
[22]
.
Our study showed that sperm counts was decreased
significantly in the lead group compared to control,
suggesting that lead reduces sperm count levels. The
administration of quercetin and omega 3 treatment
significantly improved sperm count levels, thereby reversing
the effects of lead. This corresponds to a study that showed
DHA and EPA concentrations in omega 3 corresponded
with those in spermatozoa. This proves the significant
improvement of total sperm counts and sperm concentration
in omega 3 treated group [23]. Sperm viability is the amount
of live sperm cells in the semen sample which is converted
to percentage. At least 58% of the sperm present in the
semen should be viable [24]. Sperm viability in the lead
groups was reduced compared to the control group though
not significantly. This suggests that lead as a tendency to
affect viability of sperm. However, the treatment showed no
significant difference compared to the lead group.
Morphology is one of the sperm function parameters, it
assesses sperm cells that have physical and structural
abnormalities with the head, mid piece or tail.
Morphological abnormality in greater numbers in the sperm
than 25% can lead to infertility [25]. In teratoospermia there
is less than 4% morphologically normal spermatozoa. This
study showed a significant decrease in sperm cells with
normal morphology and an increase in abnormal sperm cells
in lead group compared to the control groups. However,
treatment with quercetin and omega 3 improved the number
of normal sperms. This suggests that quercetin and omega 3
helps in sustaining the morphology of sperm cells.
A study on in vivo administration of quercetin had
documented near-normal sperm functions and morphology,
and that these effects however were most likely caused by
the inability of this com pound to ameliorate oxidative stress
and inflammation in sperm [22]. No significant alterations
were observed in FSH levels following lead administration.
Also no significant difference was observed in omega 3 and
quercetin treated groups when compared to the lead group.
Some studies indicate that dietary supplementation of omega
3 decreased serum FSH level in normal weight women but
not in obese women [26]. This could suggest why there was
no difference in the treatment group compared to the lead
group.
LH hormones showed significant reduction in the lead
group compared to the control group. Suggesting that lead
affects hormone levels of LH. No significant difference was
observed in the lead + quercetin, and lead + omega 3
treatment groups. However, the combined administration of
Lead+ quercetin and Omega 3 showed improved increase in
the LH levels. In males, LH aids in production of
testosterone. Testosterone is converted to
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ISSN No:-2456-2165
dihydrotestosterone (DHT) by 5-alpha reductase. DHT is a [7].
more potent form of testosterone and is synthesized by the
prostate gland. DHT is also responsible for the growth and
advancement of the prostate, scrotum, penis, male hair [8].
pattern, acne and baldness [27]. Factors that reduce levels of
testosterone greatly affect spermatogenesis [28, 29]. The lead
group showed significant reduction in testosterone levels
compared with the control. The levels of testosterone were
significantly increased following administration of quercetin
and omega 3 treatments. [9].
V. CONCLUSION
[10].
It can be concluded from this study that lead exposure
leads to impairment of reproductive parameters (sperm
count, viability and motility). It also caused reduction in sex
hormone (LH and testosterone) concentrations. These [11].
deleterious effects of lead on reproductive parameters were
ameliorated following omega-3 and quercetin administration
in rats. Combination of both omega-3 and quercetin
provided better ameliorative effect on total sperm count than
single administration of omega-3 or quercetin.
ACKNOWLEDGEMENTS [12].
Authors hereby acknowledge Mr. Ededet Umoh of
Physiology Department, University of Calabar, Nigeria for [13].
assisting in the authorization and collecting of blood and
semen samples used for this study. The cooperation of Dr.
Bassey Icha of Chemical Pathology Department, UCTH, [14].
Nigeria is also acknowledged for running the biochemical
assay of this research work.
 Conflict of Interest [15].
The author(s) declare(s) that there is no conflict of
interest regarding the publication of this article.
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Volume 9, Issue 2, February 2024 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/IJISRT24FEB084

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